1. Field of the Invention
The invention relates to the method of cryopreservation of biological tissues. It is particularly related to minimizing the amount of cell division for cells that will be used for the purpose of somatic cell nuclear transfer, while providing cells in which to test cell viability.
2. Background of the Invention
All documents referenced herewith are incorporated by reference. Regarding prior knowledge, cryopreservation of tissues for subsequent use in a number of applications has been reported. For example, cryopreservation of ovarian tissue is a promising technique for preserving fertility in cancer patients who are at risk of sterilization from radiation and/or chemotherapy treatment or for safeguarding reproductive potential of endangered species (Oktay et al., 1998; Abir et al., 1998). Cryopreservation of other tissue types such as skin (Sheridan et al., 1998), kidney (Sommer et al., 1999), liver (de Kanter et al., 1998), corneas (Wusteman et al., 1999), pancreatic islets (Zieger et al., 1999), and testicular tissue (Gianaroli et al., 1999) have also been reported.
The purpose of the invention is to minimize cell division until such time as cells are needed for the nuclear transfer. This method will be useful for any process in which cells need to be preserved by cryopreservation but also need to be tested for various factors. The method would provide an economic benefit by limiting the labor, supplies costs, and storage costs of generating a large amount of cells prior to the time when they are needed. The method provides a reduction in time that tissues are in the culture dish and allowed to explant cells before cryopreservation.
Cryopreservation methods include rapid vitrification (30,000xc2x0 C./minute), slow vitrification (8,000xc2x0 C./minute), rapid freezing (100xc2x0 C./minute), and slow freezing (0.5xc2x0 C./minute). The method of cryopreservation used is important due to the freezing of intracellular water. During slow cooling, water leaves the cell because of the osmotic imbalance caused by the lower concentration of water in the extracellular environment due to ice formation. The increase in solute concentration due to the decrease in water volume can be harmful. Alternatively, too much water in the inside the cell can lead to damage during warming. Cryoprotectants protect cells by diluting salt that becomes more and more concentrated as ice forms. They also stabilize membranes and proteins and reduce the intracellular ice formation temperatures. Cell survival is low at very slow and very fast cooling rates (U.S. Pat. No. 5,891,617).
Dimethylsulfoxide (DMSO) and glycerol are the most commonly used cryoprotectants. DMSO causes a depression in the freezing point and therefore increased water removal from the cell prior to freezing. DMSO generates heat when dissolved in aqueous solutions. It must be diluted and allowed to cool before addition to cells. DMSO and glycerol are usually used in a 5-10% solution in growth medium. They are not used together except for cryopreserving plant cells. Cells may be incubated in the cryoprotectant before beginning the cooling process. This is called the equilibration period. Cooling rates of 1xc2x0 C. per minute and the use of a cryoprotective agent are commonly used to protect the cells. Larger cells generally require greater control of cooling rates. Frozen cells should be maintained below xe2x88x92130xc2x0 C.
When thawing of the sample is desired, warming should occur as quickly as possible. This is generally achieved by placing the vial into 37xc2x0 C. water. The outside of the vial is disinfected before the vial is opened to protect against contamination of the sample. The cells are then transferred to fresh growth medium to decrease the concentration of the cryoprotectant. The cells can be centrifuged and the supernatant removed. The cells are then resuspended in fresh growth medium (Simione, F. P., 1998).
PCT Patent Application No. PCT/US92/00599 describes a tissue preservation method in which dissected heart valve, veins and musculoskeletal connective tissue are divided into at least two portions that are contaminated with microbes to various degrees. The group containing a lower level of contamination is exposed to an antimicrobial regimen and cryopreserved. The group containing the higher level of contamination is exposed to a second antimicrobial regimen and cryopreserved. This reference is directed to the decrease of microbial contamination of heart valves, veins and musculoskeletal connective tissue. The antimicrobial regimen lasts preferably 4 hours.
U.S. Pat. No. 5,891,617 describes cryopreservation of harvested mammalian tissues and cultured tissue equivalents in a cryoprotectant solution. Solutions contain a cell penetrating glass forming agents such as propylene glycol, ethylene glycol, and dimethylsulfoxide or non-cell penetrating glass forming agents such as high molecular weight complex carbohydrates. These solutions are diluted in a base at physiological pH. The preferred solution is 2 M glycerol in Dulbecco""s Modified Eagle""s Medium (DMEM).
U.S. Pat. No. 5,964,096 describes a method and package design for cryopreservation and storage of cultured tissue equivalents. The cryopreservation method includes immersing the tissue in cryoprotectant solution, agitating the sample, cooling to solid-liquid phase equilibrium temperature for the cryoprotectant, seeding extracellular ice, and freezing the tissue to a temperature below xe2x88x9270xc2x0 C. Cryopreserved tissue is warmed at a high rate by direct application of a warmed media or buffered solution or other heating method. Cryoprotectant is removed form the thawed tissue before use. Also described is a package that has an improved heat transfer rate, allowing for a more controlled cooling and heating process.
U.S. Pat. No. 5,328,821 describes methods and solution for the preservation of tissue. Particularly it relates to methods and solutions for preserving human tissue slices. Preservation solutions of this invention contain a sufficient amount of glucose to maintain the metabolic functions of the cells but not enough to stimulate acidosis.
U.S. Pat. No. 5,118,512 describes a process for cryopreserving bone with a cryopreservation agent and an agent to increase diffusion of the cryopreservation agent into the biological material.
U.S. Pat. No. 4,559,298 describes a method for the cryopreservation of biological materials by cooling the biological material to a vitreous state under pressure in the presence of an aqueous vitrification solution.
The present invention provides a method to both test the viability of the cells and protect cells from aging due to cell division. In addition, the present method also provides a method of obtaining a large number of cells from a single sample without exposing all of the cells to excessive cell division.
An embodiment of the present invention is a process for preserving viable tissues and cells comprising; obtaining a tissue specimen; dividing the tissue specimen into at least 2 portions; cryopreserving directly at least one portion of the tissue to minimize cell division; culturing at least one remaining portion or portions of tissue specimen in order to propagate cells and verify their viability; and cryopreserving cells obtained from tissue outgrowths or passage; and cryopreserving the cultured tissue portion or portions once the desired amount or number of cells have been obtained through outgrowth or passage. The use of the phrase xe2x80x9coutgrowth or passagexe2x80x9d means xe2x80x9coutgrowth and/or passage,xe2x80x9d since the term xe2x80x9corxe2x80x9d is not intended to be exclusive. The term xe2x80x9cdirectlyxe2x80x9d means that an action is taken without performing any substantive process prior to taking that action, for example, xe2x80x9ccryopreserving directlyxe2x80x9d means not doing any other step than those to achieve cryopreservation. It is contemplated that the tissue may be divided into 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more portions, and that at least one portion is cryopreserved directly or prior to cell division taking place.
In another embodiment, the cryopreservation of cultured tissue occurs simultaneously with the cryopreservation of the cells. The term xe2x80x9csimultaneouslyxe2x80x9d means at the same time, that is, less than an hour within each other. In yet another embodiment, the cryopreservation of cultured tissue occurs after the cryopreservation of the cells. The cryopreservation of cultured tissue may occur more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6 months after the cryopreservation of the cells obtained from tissue outgrowths or passage.
In another embodiment, the process for preserving viable tissues and cells is for use in nuclear transfer. The tissue preserved may be that of a domestic, exotic, or food animal. In a further embodiment it is that of a mammal. More specifically, the tissue may be that of a dog, cat, horse, sheep, goat, or cow and most specifically that of a dog or cat.
In still another embodiment, the process for preserving viable tissues and cells may include an embodiment where tissue portions constituting the directly cryopreserved tissue and cultured tissue are less than or about 1 millimeter3 pieces or portions.
In another embodiment, the process for preserving viable tissues and cells may include the cells or tissues to be cryopreserved being placed in a medium comprising less than or equal to 95% Basal Medium Eagle (BME), Dulbecco""s Modified Eagles Medium (DME), Nutrient Mixture Ham""s F-10, Nutrient Mixture Ham""s F-12, Dulbecco""s Modified Eagles Medium Nutrient Mixture F-12 Ham (DME/F12 1:1 mixture), L-15 Medium Leibovitz, McCoy""s 5A Medium, Medium 199, Minimum Essential Medium Eagle (MEM), RPMI-1640 Medium, or Waymouth""s Medium. More specifically, the medium may further comprise from about 5-20% Fetal Bovine Serum, at least 100 Units/milliliter penicillin, at least 0.1 milligrams/milliliter streptomycin, at least 0.25 micrograms/milliliter amphotericin B, and 10%-20% dimethylsulfoxide.
In another embodiment, the process for preserving viable tissues and cells may include placing the tissues in said medium for about 10 minutes before cryopreservation.
In another embodiment, the process for preserving the cells may include placement in said medium directly before cryopreservation.
In another embodiment, the tissues or cells are frozen at a rate of about xe2x88x921xc2x0 C./min. In yet another embodiment, the tissues or cells may be placed in an xe2x88x9280xc2x0 C. freezer overnight and transferred into liquid nitrogen the next day. It is contemplated that the xe2x80x9cnext dayxe2x80x9d refers to an amount of time greater than 8 hours. In still another embodiment, after initial cryopreservation and before thawing is desired, the temperature of said tissue or cells may not exceed xe2x88x9260xc2x0 C.
In another embodiment, the process for preserving viable tissues and cells may include placing cells or tissues to be cultured in a medium comprising less than or equal to 95% Basal Medium Eagle (BME), Dulbecco""s Modified Eagles Medium (DME), Nutrient Mixture Ham""s F-10, Nutrient Mixture Ham""s F-12, Dulbecco""s Modified Eagles Medium Nutrient Mixture F-12 Ham (DME/F12 1:1 mixture), L-15 Medium Leibovitz, McCoy""s 5A Medium, Medium 199, Minimum Essential Medium Eagle, RPMI-1640 Medium, or Waymouth""s Medium. More specifically, the medium may further comprise from about 5-20% Fetal Bovine Serum, at least 100 Units/milliliter penicillin, at least 0.1 milligrams/milliliter streptomycin, and at least 0.25 micrograms/milliliter amphotericin B. The medium may comprise about 10% Fetal Bovine Serum, at least 100 Units/milliliter penicillin, at least 0.1 milligrams/milliliter streptomycin, and at least 0.25 micrograms/milliliter amphotericin B.
In another embodiment, a process for tissue decontamination may be included in the preserving of viable tissues and cells comprising: incubating tissues in an about 1:30 chlorhexidine solution for not more than about 3 minutes; agitating tissues during the incubation period; placing tissues into a medium comprising Ham""s F-12 and at least 600 Units/milliliter penicillin, at least 5 milligrams/milliliter streptomycin, and at least 1.5 micrograms/milliliter amphotericin B.; soaking tissues for about 40 minutes at about 4xc2x0 C.; and proceeding with tissue isolation procedures, for example, as described herein.
In yet another embodiment, an apparatus for shipping tissue specimens to be preserved by the process above may be used with the apparatus comprising sterile tubes containing a sterile media storage solution, an insulated shipping container, and ice packs. The apparatus may further include sterile media comprising less than or equal to 95% Basal Medium Eagle (BME), Dulbecco""s Modified Eagles Medium (DME), Nutrient Mixture Ham""s F-10, Nutrient Mixture Ham""s F-12, Dulbecco""s Modified Eagles Medium Nutrient Mixture F-12 Ham (DME/F12 1:1 mixture), L-15 Medium Leibovitz, McCoy""s 5A Medium, Medium 199, Minimum Essential Medium Eagle, RPMI-1640 Medium, or Waymouth""s Medium. More specifically, the sterile media may further comprises from about 5-20% Fetal Bovine Serum, at least 100 Units/milliliter penicillin, at least 0.1 milligrams/milliliter streptomycin, and at least 0.25 micrograms/milliliter amphotericin B.
In still another embodiment, a method for maintaining tissue viability while transferring biological specimens to be preserved by the process above, may comprise receiving a storage box, refrigerating tubes of sterile storage solution, placing tissues in the tubes, and shipping to destination overnight in said storage box containing frozen ice packs.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.